Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ Treg

Authors

  • Ling Lu,

    1. Division of Rheumatology, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
    2. Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, P. R. China
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    • These authors contributed equally to this work.

  • Jilin Ma,

    1. Division of Rheumatology, Immunology and Nephrology, Zhejiang Traditional Chinese Medicine and Western Medicine Hospital, Hangzhou, P. R. China
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    • These authors contributed equally to this work.

  • Xuehao Wang,

    Corresponding author
    1. Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, P. R. China
    • Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, P. R. China
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  • Julie Wang,

    1. Division of Rheumatology, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
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  • Feng Zhang,

    1. Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, P. R. China
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  • Jiangning Yu,

    1. Division of Rheumatology, Immunology and Nephrology, Zhejiang Traditional Chinese Medicine and Western Medicine Hospital, Hangzhou, P. R. China
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  • Ge He,

    1. Division of Rheumatology, Immunology and Nephrology, Zhejiang Traditional Chinese Medicine and Western Medicine Hospital, Hangzhou, P. R. China
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  • Bing Xu,

    1. Developmental Biology Program, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA
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  • David D Brand,

    1. Research Service, Veterans Affairs Medical Center, Memphis, TN, USA
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  • David A Horwitz,

    1. Division of Rheumatology, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
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  • Wei Shi,

    1. Developmental Biology Program, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA
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  • Song Guo Zheng

    Corresponding author
    1. Division of Rheumatology, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
    • 2011 Zonal Avenue, Hoffman Medical Research Building, Room 710A, Los Angeles, CA 90033, USA Fax: +1-323-442-2874
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Abstract

TGF-β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF-β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and -4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-β to induce naïve CD4+CD25 cells to express Foxp3; however, BMP-2/4 and TGF-β have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-β. Data further advocate that TGF-β is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-β indicates that the simultaneous manipulation of TGF-β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.

Introduction

A subset of CD4+ Treg, expressing the IL-2 receptor α chain (CD25) and the nuclear transcription factor Foxp3, plays an important role in the maintenance of immune homeostasis and prevention of autoimmunity 1, 2. These cells comprise thymus-derived, naturally occurring CD4+ Treg (nTreg) and those that can be induced in the periphery (induced Treg, iTreg) 3. Although Treg subsets may have different developmental mechanisms, they share similar phenotypes and suppressive activities 4. These Treg populations may have a synergistic action or have different targets that maintain immune homeostasis 5.

TGF-β is essential for the induction of Foxp3 expression and for suppressive activity in iTreg, as well as the maintenance of both Foxp3+ nTreg and iTreg 6–11. It is still unclear whether other molecules except TGF-β also promote the development of Foxp3+ iTreg. In addition, the TGF-β signaling pathway is not essential for the development of nTreg since both TGF-β and TGF-β receptor II KO mice express Foxp3+ nTreg in the thymus 10, 12, 13. It has been suggested that other member(s) of the TGF-β superfamily such as bone morphogenetic proteins (BMP) might substitute for TGF-β during the development of Treg 14.

BMP, with more than 20 family members, have been shown to regulate many fundamental biological processes including cell proliferation, differentiation, apoptosis, migration, and adhesion 15. Furthermore, BMP are involved in the development of almost all tissues and organs, including thymocytes and hematopoietic stem cells 16. Recent studies have revealed that transgenic expression of the BMP antagonist Noggin in thymic epithelial cells results in the development of a smaller-sized thymus 17. In a manner similar to that of TGF-β, treatment of purified human CD34+CD38 cells with BMP-2, -4, or -7 not only affected their own proliferation and differentiation, but exerted a direct effect on human stem cell survival as well 18. As extracellular growth factors, BMP bind to heteromeric complexes of BMP serine/threonine kinase type I (ALK2, ALK3, and ALK6) and type II receptors 19, 20. Upon ligand-induced aggregation of the receptors, constitutively activated BMP type II receptor kinase phosphorylates and activates the type I receptor, which subsequently recognizes and phosphorylates receptor-bound BMP-specific Smad proteins (Smad1, 5, and 8) on the carboxyl terminal SSXS motif. These Smads dissociate from the receptors, form complexes with Smad4, translocate into the nucleus, bind to BMP responsive element, and act as transcriptional co-modulators to induce or repress BMP-target gene expression 21. BMP-2 and -4 are two common forms and typical representatives of BMP that affect the T-cell development in the thymus 22. CD4+ cells have been found to express BMP receptors I and II and treatment of BMP induced CD4+ cells to express phosphorylation of Smad1, 5, and 8 23.

It still remains unclear whether BMP affect the development of Foxp3+ Treg. As BMP-2−/−, BMP-4−/−, and their downstream molecule Smad5−/− mice all die at mid-gestation due to multiple embryonic and extraembryonic defects 24, 25, it is difficult to determine their role in the development of nTreg. To learn the role of BMP signaling in the induction of iTreg, we have demonstrated here that both BMP-2 and -4 are unable to substitute for TGF-β in the differentiation of Foxp3+ iTreg. Addition of BMP-2 or -4 to TCR-activated CD4+ cells failed to induce the expression of Foxp3 and the development of suppressive activity. However, the addition of BMP-2 and -4 significantly increased the ability of TGF-β to promote the generation of Foxp3+ iTreg, and this synergistic effect was also dependent upon TGF-β receptor signaling, as well as ERK and/or JNK MAPK pathways. Moreover, lack of BMP receptor signaling did not impair the TGF-β-mediated induction of Foxp3+ iTreg. Our results demonstrate that TGF-β superfamily members, BMP and TGF-β, each may play a distinct role in modulating Treg development.

Results

BMP-2 and -4 are unable to induce Foxp3+ iTreg in the absence of TGF-β

We and others have reported previously that TGF-β can induce TCR-activated naïve CD4+ CD25 cells to become CD4+CD25+ Foxp3+ iTreg 6–9. Because BMP-2 and -4 both belong to the TGF-β superfamily and exert similar effects on the development of early thymocytes and peripheral lymphocytes as TGF-β 17, 18, we initially sought to determine if these members could mediate the induction of Foxp3+ in a manner similar to that of TGF-β. Unexpectedly, we found that the addition of BMP-2 or -4 to TCR-activated naïve CD4+CD25 cells did not induce Foxp3 expression (Fig. 1A–C). In addition, the resultant CD4+CD25+ cells did not exhibit an anergic status, one of the hallmark features of Treg (Fig. 2A). Accordingly, they failed to suppress T-cell proliferation (Fig. 2B and C). Conversely, addition of TGF-β1 induced the expected Foxp3 expression and these cells displayed typical anergic status and potent suppressive activities (Fig. 1A–C, Fig. 2). Further experiments with time-course study showed that BMP-2 or -4 alone was unable to induce Foxp3 expression over a week-long culture period (Fig. 1D), despite providing a wide variety of physiologic doses of BMP-2 or -4 (Fig. 1E).

Figure 1.

BMP-2 and -4 fail to induce Foxp3 expression on CD4+ cells. (A) Splenic naïve CD4+CD25 cells isolated from C57BL/6 mice were stimulated with anti-CD3/CD28-coated beads (1:5) in the presence of IL-2 (20 U/mL) and TGF-β (2 ng/mL), or BMP-2 (10 ng/mL), or BMP-4 (10 ng/mL) for 4 days. Intracellular Foxp3 protein expression was analyzed by flow cytometry. Data show mean±SEM of five independent experiments. (B) Representative histogram of experiments described in (A). The percentage of CD25+ cells expressing Foxp3 is shown in the upper right of the histogram. (C) Foxp3 mRNA expression as determined by real-time PCR after normalization of β-actin. Data show mean±SEM of four independent experiments. (D) Foxp3 and CD25 expression over time using a similar experimental design as in (A). (E) Dose effect of BMP-2 and -4 on the induction of Foxp3 expression. Data from (D and E) show mean±SEM of four independent experiments.

Figure 2.

BMP-2/4-primed CD4+ cells are unable to display the phenotypes and function of suppressor cells. (A) BMP2/4- or TGF-β-primed CD4+ cells were induced as described in Fig. 1. These cells were restimulated with anti-CD3 (0.25 μg/mL) in the presence of APC (3000-radγ irradiation) for 3 days and proliferation was assayed by 3H-thymidine incorporation. (B) CD4+CD25 T cells labeled with CFSE were stimulated with soluble anti-CD3 and APC, or with BMP-2-, -4-, TGF-β-primed CD4+ or control (Med) cells for 4 days. The ratio of conditioned cells to responder T cells is1:4. The % suppression was calculated as previously reported 11. (C). Cytometric data representative of four independent experiments in (B). (D and F). C57BL/6 mice received i.p. injections of BMP-2 (0.5 mg/mouse), BMP-4 (0.5 mg/mouse) or control PBS every other day for 10 days. The frequency (D) and suppressive activities (E) of CD4+CD25+Foxp3+ cells in thymus, spleen, and blood in these mice were determined at day 12 after administration using flow cytometry and a standard in vitro suppressive assay as above. Each group includes four mice and values indicate mean±SEM. Note that there is no significant difference between mice receiving BMP-2/4 or PBS. (F) Naïve CD4+ cells isolated from C57BL/6 mice were treated with BMP-2/4 with or without TGF-β for 2 h and the Smad1 and phosphorylated Smad1 expression was determined by western blot. Data are representative of three independent experiments.

We next provided further evidence that treatment of BMP-2 or -4 in mice does not change CD4+Foxp3+ cell frequency and function in vivo. As shown in Fig. 2D, we observed that percentages and total numbers (data not shown) of CD4+CD25+Foxp3+ cells were not changed at day 10 following i.p. injection of BMP-2 and -4 compared with mice that received a similar volume of PBS. We observed a markedly increased expression of phosphorylated Smad1 in T cells at 6–24 h post-injection of BMP-2/4 (data not shown). It has been reported that BMP signal through serine/threonine kinase receptors and activate downstream molecules of BMP receptors such as Smad1, 5, and 8 21, 23. We also compared the suppressive activity of CD4+CD25+ cells on T-cell responses; we found that CD4+CD25+ cells sorted from mice treated with BMP or PBS displayed similar suppressive activities on T-cell proliferation (Fig. 2E). Taken together, these data indicate that BMP do not affect the differentiation of CD4+Foxp3+Treg in vitro and in vivo.

Further experiments excluded the possibility that failure of iTreg induction by BMP is due to lack of endogenous BMP signaling activity in naïve CD4+CD25 cells. As reported before, some CD4+ cell subsets express BMP receptors I and II 23. We observed that naïve CD4+CD25 cells expressed BMP receptor I (data not shown). Moreover, BMP-2- or -4-primed naïve CD4+CD25 cells exhibited a distinct expression of phosphorylated Smad1 and addition of TGF-β did not alter the level of phosphorylated Smad1 expression induced by BMP-2 or -4 (Fig. 2F).

Induction of Foxp3+ by TGF-β is independent of BMP receptor signal

With evidence suggesting that BMP-2 and -4 alone do not promote the differentiation of Foxp3+ iTreg, we next explored whether BMP signaling affects the induction of Foxp3+ iTreg by TGF-β. It has been reported that Noggin is a BMP antagonist that inhibits the binding of BMP to their cognate receptor 26. In this study, we observed that addition of Noggin did not alter the TGF-β-induced Foxp3 expression (Fig. 3A) and did not affect their suppressive activities (Fig. 3B). In fact, we have observed that addition of Noggin significantly decreased the expression of phosphorylated Smad1 induced by BMP-4 (Fig. 3C). These results suggest that the endogenous BMP signal pathway alone plays no role in the differentiation of Foxp3+ iTreg.

Figure 3.

Induction of Foxp3+ by TGF-β is independent of BMP receptor signal. (A) Foxp3 expression was determined in CD4+ cells treated with medium, TGF-β, and both TGF-β and Noggin treated CD4+ cells. Data are representative of three independent experiments. (B) The suppressive activity of these cells was analyzed by a standard suppressive assay as described in Fig. 1B. (C) The effects of Noggin on the Smad1 activation of BMP-4 on CD4+ cells. Naïve CD4+ cells were stimulated with BMP-4 with or without Noggin for 2 h and Smad1 and phosphorylated Smad1 expression were determined by western blot. Experiments were repeated twice with similar results.

Synergistic effect of exogenous BMP-2/4 and TGF-β on the induction of Foxp3+ iTreg

Unlike TGF-β, BMP-2/4 alone did not induce the differentiation of Foxp3+ iTreg. However, addition of exogenous BMP-2 or -4 significantly increased the ability of TGF-β to promote the development of iTreg. As shown in Fig. 4A, naïve CD4+CD25 cells treated with BMP-2 or -4 in combination with TGF-β produced significantly higher Foxp3+ cell numbers than those treated with TGF-β alone in vitro. Moreover, CD4+ cells treated with the combination of both BMP-2/4 and TGF-β displayed more potent suppressive activity against T-cell proliferation in vitro than cells treated with TGF-β alone (Fig. 4B). In an animal model of chronic graft versus host disease (cGVHD) with a typical lupus-like syndrome, we found that co-transfer of BMP-4/TGF-β-treated CD4+ cells and pathogenic DBA/2 splenocytes more efficiently suppressed anti-DNA production than that of TGF-β-treated CD4+ and DBA/2 splenocytes in (DBA/2xC57BL/6) F1 mice (Fig. 4C).

Figure 4.

BMP-2/4 enhances TGF-β induced development of CD4+Foxp3+ Treg. (A) Effect of BMP-2/4 on the Foxp3 expression on CD4+ cells. Suppressive activities of Treg subsets against T-cell proliferation in vitro (B) and anti-dsDNA production in cGVHD with a lupus syndrome model (n=four mice each group) (C) were determined, described in the Materials and methods. Values are the mean±SEM of three separate experiments (B). (D) The frequency of CD4+CD25+Foxp3+ cells in spleen, LN, and gut of mice receiving PBS, or BMP-4, or TsA, or BMP-4+TsA at day 12 after treatment. Values indicate Mean±SEM (left panel) of three mice in each group and representative of these experiments (right panel). Experiment was repeated with similar results. (E) Splenic CD4+CD25+ cells were sorted by FACSVantage and their suppressive activity against T-cell proliferation in vitro was analyzed as (A). (F and G) CD4+Foxp3+ cells were induced for 2 days as in Fig. 1 and labeled with CFSE, and stimulated with either IL-2 (40 U/mL), or BMP-2 (10 ng/mL), or BMP-4 (10 ng/mL) for additional 4 days. Total Foxp3+ cells were counted every 2 days (F) and Foxp3+ cell proliferation levels were analyzed by the dilution of CFSE dye on day 6 (G). Values indicate Mean±SEM of three separate experiments (F) and the data presented are representative of these experiments (G). (H) Bcl-2 mRNA expression was determined by quantitative RT-PCR. Data show mean±SEM of triplicate samples and experiment was performed with similar results.

We further investigated whether BMP and TGF-β exhibited a synergistic role in the induction of Foxp3+ Treg in vivo. As TGF-β has a very short half-life in vivo, it is not practical to evaluate synergistic effects of BMP and TGF-β following injection in vivo. However, recent studies indicated that trichostatin A (TsA), one of the histone deacetylase inhibitors, is able to promote induction and conversion of CD4+CD25 cells to Foxp3+ Treg in vivo27, 28; we confirmed this result and found that the combination of TsA and BMP-4 administration resulted in significantly increased CD4+CD25+Foxp3+ cells in vivo than TsA treatment alone, particularly in the gut (Fig. 4D). Moreover, splenic CD4+CD25+ cells sorted from BMP-4- and TsA-treated mice displayed enhanced suppressive activity against T-cell proliferation compared with mice treated with TsA or BMP-4 alone (Fig. 4E). We also showed BMP-4 alone did not significantly increase Foxp3+ cell frequency in gut (Fig. 4D) as well as other organs (Fig. 2D). Co-injection of BMP-2 and TsA had a similar result (data not shown). These data further support the synergistic effect of BMP on TGF-β-induced iTreg generation.

We next investigated the mechanisms by which BMP promote Foxp3+ cell production induced by TGF-β. We previously have reported that induction of Foxp3 by TGF-β occurs only when this cytokine was added to cultures within 24 h of TCR stimulation 11. We now induced Foxp3+ cells for 2 days first and then labeled them with CFSE. These cells were extensively washed and exogenous IL-2, BMP-2, or BMP-4 were added to cultures to learn whether BMP-2 or -4 can expand Foxp3+ cells in a manner like IL-2. Unlike IL-2, neither BMP-2 nor -4 significantly expanded Foxp3+ cells that had been generated by TGF-β (Fig. 4F and G). Additionally, the addition of BMP-2/-4 to TGF-β did not increase the expression of Bcl-2 mRNA (Fig. 4H), one of key anti-apoptosis genes, suggesting that BMP promote Foxp3+ cells through increasing conversion rather than expanding or prolonging the survival of these cells.

We also documented that the addition of the BMP antagonist Noggin significantly abolished the enhanced Foxp3 expression induced by BMP-2/4+TGF-β (Fig. 5A). The synergistic effect of BMP-2/4 and TGF-β is also fully dependent upon TGF-β receptor signaling since addition of TGF-β receptor I (ALK5) inhibitor almost completely suppressed Foxp3 expression and this effect also disappeared when naïve CD4+CD25 cells from WT mice were substituted from TGF-β receptor II KO mice (Fig. 5B).

Figure 5.

Noggin blocks the BMP2/4 enhancement of TGF-β-induced Foxp3 expression. Naïve CD4+CD25 cells isolated from C57BL/6 WT and TGF-β receptor II KO mice were stimulated as described in Fig. 1, in some cultures, Noggin (100 ng/mL) or ALK5 (TGF-β RI) inhibitor (5 μM), or DMSO control was added to cultures and Foxp3 expression was determined as above. Values indicate the mean±SEM of three independent experiments.

It seems unlikely that BMP-2 or -4 simply enhances the role of TGF-β in the induction of iTreg through activation of TGF-β receptor-related Smad molecules since BMP-2 or -4 activates Smad1, 5, 8 whereas TGF-β activates Smad2 and 3. However, it should be noted that the Smad-independent signaling molecule, P38, one of MAPK could be involved in the development of Foxp3+ iTreg induced by TGF-β 29. In fact, we have observed both Smad and non-Smad signaling pathways are required for TGF-β to induce Foxp3+ iTreg (Lu and Zheng; unpublished data). Thus, we next tested whether BMP-2 or -4 affects the MAPK expression. As shown in Fig. 6A, TCR-activated naïve CD4+CD25 cells cultured in the presence of BMP-4 did not induce p-Smad2/3 expression and did not enhance the p-Smad2/3 expression induced by TGF-β; however, they did affect the expression of phosphorylated ERK1/2 and phosphorylated JNK expression although they had little influence on phosphorylated P-38 expression. As shown in Fig. 6B, either BMP-4- or TGF-β-treated CD4+ cells expressed activated JNK in 12 h and rapidly disappeared in 24 h following TCR stimulation. Interestingly, both BMP-4 and TGF-β together began to induce activation of JNK in 4 h and significantly increased the expression of activated JNK in 12 h and somewhat maintained its expression until 24 h after TCR stimulation. Unlike JNK activation, BMP-4 treatment alone induced lower level of activated ERK in 24 h, TGF-β treatment alone slightly increased ERK activation, interestingly, combination of both resulted in a marked induction of phosphorylated ERK in 12−24 h although this expression significantly decreased in 48 h after TCR stimulation (Fig. 6C). Time-course experiments revealed that ERK1/2 or JNK activation appears to be in the early stage of TCR/TGF-β stimulated CD4+ cells, further suggesting that BMP promote Foxp3+ cell conversion rather than expansion (Fig. 6B and C).

Figure 6.

BMP-2/4 on enhancement of TGF-β-induced CD4+Foxp3+ Treg development involves MAPK activation. (A) Naïve CD4+ cells were TCR (anti-CD3/CD28 coated bead 1:5) stimulated with or without BMP-4±TGF-β for 16 h. Activated Smad2/3 expression was determined by western blot. These cells were stimulated with TCR in the presence of BMP-4, TGF-β, or both (BMP-4+TGF-β) in various time-points as indicated and the levels of activated JNK (B) and ERK (C) were determined by western blot. (D) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4+ TGF-β in the presence or absence of MAPK inhibitors. (E) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4±TGF-β in ERK1 KO, JNK2 KO, and littermate control mice for 4 days. (F) Foxp3 expression on CD4+ cells stimulated with TCR with BMP-2/4±TGF-β in IL-10 KO and littermate control mice for 4 days. In some experiments with WT control mice, anti-IL-10, anti-IL-10R, or control IgG were added to cultures. Data show mean±SEM of three separate experiments or represent at least three independent experiments.

Of interest, when ERK or JNK inhibitors (PD 98059 or SP600125) but not p-38 inhibitor (SB203580) was added to cultures, the enhancement of Foxp3 expression by BMP-4 in the presence of TGF-β was completely abolished (Fig. 6D). This suppression cannot be explained by the non-specific suppression since the CD25 expression by CD4+ cell and total viable CD4+ cell numbers were comparable between groups treated with MAPK inhibitors or with DMSO control (data not shown). It is noted that Foxp3 expression by BMP-4/TGF-β was even lower than that by TGF-β alone when ERK inhibitor was added to cultures, suggesting that ERK activation also involves the development of Foxp3+ Treg induced by TGF-β. To further exclude the possibility that ERK or JNK inhibitor non-specifically suppressed T-cell activation and interfered with Foxp3+ cell development, we have also observed the role of MAPK pathways in Foxp3+ iTreg promotion by BMP using ERK1 and JNK2 KO mice. As shown in Fig. 6E, deficiency of ERK1 or JNK2 completely abrogated the enhanced Foxp3 production by BMP-4 in the TGF-β-primed CD4+ cells although these deficiencies did not interfere with CD25 expression by TCR-activated CD4+ cells, suggesting that the synergistic effect of BMP and TGF-β depends upon either ERK or JNK MAPK pathway.

The ERK pathway is important for the production and function of IL-10 and IL-10 induces IL-10-producing type I Treg development 30. We sought to determine whether IL-10 plays a role in the synergistic role of BMP and TGF-β in the Foxp3+ iTreg induction. As shown in Fig. 6F, addition of IL-10 to the TGF-β did not significantly increase Foxp3 production. Neutralization of either IL-10 or IL-10 receptor with respective antibodies did not alter the enhanced Foxp3+ cell induction by a combination of BMP and TGF-β. Moreover, BMP similarly promoted Foxp3+ cell production in both WT and IL-10 KO mice, suggesting that IL-10 is redundant towards the synergistic role of BMP and TGF-β in Foxp3+ cell development. Collectively, these results indicate that BMP-induced ERK1/2 and/or JNK might either affect activated Smad molecules induced by TGF-β or other unknown molecules that then lead to the synergistic effect between TGF-β and BMP on the differentiation of Foxp3+ iTreg.

Discussion

Although both TGF-β and BMP similarly regulate the development of thymocytes and human hematopoietic cells 17, 18, we show here that the endogenous BMP signaling pathway is not essential for the induction of Foxp3+ iTreg. The addition of exogenous BMP-2/4 alone to TCR-activated CD4+CD25 cells fails to induce Foxp3 expression and confers immune suppressive activity. TGF-β is still capable of inducing Foxp3+ expression on naïve CD4+CD25 cells by treatment with the BMP antagonist Noggin. Although the addition of BMP-2/4 to TGF-β significantly increases the ability of the latter to induce iTreg development, this synergistic effect is also TGF-β-dependent since blockade of TGF-β receptor signaling abrogates Foxp3 induction. Both BMP and TGF-β together enhance the expression of the activated ERK and JNK MAPK to promote Foxp3+ iTreg development. These findings indicate that TGF-β rather than BMP members play an essential role in the induction of Foxp3+ Treg despite both belonging to the TGF-β superfamily.

We and others have provided considerable evidence that TGF-β induces the differentiation of iTreg that express Foxp3 and suppress T-cell immune response 6–9. Adoptive transfer of iTreg can control many autoimmune diseases and protect heart transplantation allograft survival 31, 32. However, other stimulation without TGF-β also seems to be able to induce Foxp3+ expression 33. As BMP belongs to the TGF-β superfamily, we investigated whether BMP might substitute for TGF-β in this capacity. Our study demonstrates that both endogenous and exogenous BMP-2 and -4 are not essential for the induction of Foxp3+ expression and suppressive capacity in these cells.

CD4+ cells express BMP receptors, and treatment of the cells with BMP-2/4 activates downstream Smad1/5/8 by protein phosphorylation, suggesting that the failure of Foxp3+ iTreg induction is not due to the inability of CD4+ cells to respond to BMP-2 or -4. Additionally, the synergistic action of exogenous BMP-2 or -4 with TGF-β on iTreg induction also indicates that CD4+ T cells do indeed respond to BMP-2 or -4.

The synergistic action of exogenous BMP and TGF-β on iTreg induction could be explained by promoting the Foxp3+ conversion from non-Treg, or expanding the Foxp3+ cells and prolonging the survival of Foxp3+ cells that had been induced by TGF-β. We have previously found that TGF-β mainly converts Foxp3+ cells in the first 24 h of TCR activation and TGF-β no longer exerts this conversion after 48 h. With this system, we now observed that BMP promotes Foxp3+ cells only when BMP were added to cultures on day 0. Unlike IL-2, BMP did not expand Foxp3+ cells when they were added to cultures 48 h after the Foxp3+ cells had been generated by TGF-β. It is known that TGF-β protects T cells from apoptosis; however, addition of BMP to TGF-β-treated cells did not enhance expression of the anti-apoptosis gene, Bcl-2. These results indicate that BMP mainly promote the ability of TGF-β to convert Foxp3+ cells.

We have concluded that this synergism is an effect of BMP-2 or -4 on TGF-β, but not TGF-β on BMP. It seems unlikely that BMP-2 or -4 activates the Smad molecules of TGF-β signal downstream (Smad2 and 3) since BMP-2 or -4 usually activates Smad1, 5, and 8. We have confirmed that CD4+ cells treated with BMP did not induce Smad2 and 3 activation. We recently found that the induction of Foxp3+ iTreg by TGF-β requires multiple pathways, and that both Smad and non-Smad pathways contribute to this induction (our unpublished observation). In this regard, BMP-2 or -4 might act through the activation of P38, ERK, and/or JNK, thus subsequently enhancing the effect of TGF-β on the induction of Foxp3+ iTreg. It has been reported that in addition to Smad-dependent signaling and transcriptional regulation, the activated receptor complex of BMP/BMP receptor also activates non-Smad signaling pathways, such as MAPK and TAK1/MEKK1, which leads to phosphorylation of ERK and p-38 in Jurkat TAg cells or non-T cells 17, 34. In this study, we observed that BMP-2 or -4 induced the activation of ERK and JNK and addition of ERK or JNK inhibitors suppressed the effects of BMP on Foxp3 expression on CD4+ cells induced by TGF-β although it did not significantly affect the p-38 activation. Time-course experiments reveal that activation of ERK and JNK appears to take place in the early stages of TCR stimulation; it is consistent with previous report that TGF-β induces CTLA-4 upregulation in first day after TCR stimulation and this early upregulation is necessary for Foxp3+ iTreg conversion 35. We further support the importance of MAPK pathways in synergistic effects of BMP and TGF-β by using ERK1 and JNK2 KO mice. Although ERK activation is important for the IL-10 production and function 30, our current study indicates that IL-10 is redundant in the synergistic effect of BMP and TGF-β in the Foxp3+ cell induction. Thus, evidence established in the current study also raise the possibility that MAPK, particularly ERK and/or JNK, activation could possibly involve crosstalk with activated Smad molecules induced by TGF-β, eventually leading to the synergistic effect on the induction of Foxp3+ iTreg.

In addition to BMP, activin-A also belongs to the TGF-β superfamily. While we prepared this manuscript, Huber et al. just reported that activin-A can convert non-Treg to Foxp3+ Treg and both actvin-A and TGF-β have a synergistic effect on the induction of Foxp3+ Treg 36. Because both Activin-A and TGF-β similarly activate Smad2 37, it is likely that activin-A can, at least in part, substitute TGF-β for the differentiation of iTreg since we have observed Smad2 had a partial role in the regulation of Foxp3+ iTreg development (Zhou and Zheng, unpublished observation).

Our findings further demonstrate that TGF-β and its receptor signaling pathways are key factors in the development of Foxp3+ iTreg. In addition, the synergistic role between BMP and TGF-β indicates that manipulation of TGF-β signaling might have a considerable impact on therapeutic applications involving iTreg through improving iTreg purity and yield.

Materials and methods

Mice

C57BL/6, IL-10 KO and JNK2 KO mice were purchased from Jackson Laboratory (Bar harbor, ME, USA), Floxed TGF-β receptor II mice were provided by Dr. Harold Moses at Vanderbilt University 38. ERK1 KO mice were provided by Dr. Gary Landreth at Case Western Reserve University. All mice were maintained in specific pathogen-free conditions according to the guidelines of the University of Southern California or Children's Hospital Los Angeles. All animals were treated according to National Institute of Health guidelines for the use of experimental animals with the approval of the University of Southern California Committee for the Use and Care of Animals.

Antibodies and reagents

Anti-CD4 (L3T4), anti-CD8 (Ly-2), and anti-CD25 (PC61.5) were purchased from eBioscience, (San Diego, CA, USA) anti-Foxp3 (150D) was purchased from Biolegend (San Diego, CA, USA). Anti-Smad1, Anti-phospho-Smad1Ser463/465, anti-phospho-Smad2/3Ser423/425, anti-phospho-P38Thr180Tyr182, anti-phospho-ENKThr202Tyr204, and anti-phospho-JNKThr183/Tyr185 were purchased from Cell Signaling Technologies (Danvers, MA, USA). ALK-5 inhibitor, TsA, and CFSE were purchased from Sigma-Aldrich. BMP-4, IL-2, and TGF-β were purchased from R&D Systems (Minneapolis, MN, USA). BMP-2 and Noggin were gifts from Dr. Yuanping Han at the University of Southern California. Anti-CD3 and anti-CD28 coated beads were purchased from Invitrogen Life Technologies (San Diego, CA, USA). AIM-V serum-free medium (Invitrogen Life Technologies, San Diego, CA, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES (all from Invitrogen Life Technologies) was used for the generation of CD4+ Treg or control cells. RPMI 1640 medium supplemented as just described with 10% heat-inactivated FBS (HyClone, Logan, UT, USA) was used for all other cultures.

Cell isolation and culture

T cells were prepared from LN and spleen cells by collecting nylon wool column non-adherent cells as previously described 31. CD4+ T cells were isolated by negative selection. Briefly, T cells were labeled with PE-conjugated anti-CD8, anti-CD11b, and anti-B220 mAb, incubated with anti-PE magnetic beads, and loaded onto MACS separation columns (Miltenyi Biotec, Auburn, CA, USA). The CD4+ cells were further labeled with FITC-conjugated anti-CD25 mAb, and CD4+CD25 cells were obtained by positive selection using MACS beads (purity >98%).

Assessment of Treg activity in vitro and in vivo

To generate Treg, naïve CD4+CD25 cells from either WT mice or gene KO mice as indicated were stimulated with anti-CD3/CD28 coated beads with or without TGF-β (2 ng/mL) in the presence of IL-2 (20 U/mL) in AIM-V serum-free medium. BMP-2 or -4 or Noggin was also added to selected cultures. Various doses of CD4+ regulatory cells (CD4TGF-β) or control CD4+ cells (CD4med) were added to fresh T cells that were activated with anti-CD3 (0.25 μg/mL) and in the presence of irradiated APC. Proliferation was assayed by CFSE dilution assay or Thymidine [H3] incorporation assay as previously described 11, and the inhibition of cycling T cells was assessed. To evaluate the suppressive effect of Treg subsets in vivo, a cGVHD with a typical lupus syndrome was induced by adoptive transfer of 80 million of DBA/2 splenocytes into (DBA/2xC57BL/6) F1 mice. In total, 5 million of CD4+ control (CD4+ cells activated with TCR without TGF-β), CD4TGF-β, or CD4BMP-4+TGF-β cells were co-transferred with pathogenic DBA/2 splenocytes into F1 mice and anti-dsDNA was measured with an ELISA every 2 wk post-transfer as before 31.

BMP and TsA administration

C57BL/6 mice was i.p. administered with either TsA (1 mg/kg) each day for 7 days, and/or BMP-2 (0.5 mg/mouse), BMP-4 (0.5 mg/mouse) or control PBS every alternate days for 10 days. The frequency of CD4+CD25+Foxp3+ cells in thymus, spleen, blood, and gut at 12 days after treatment were determined by flow cytometry. Splenic CD4+CD25+ cells in various groups of mice were sorted by FACSVantage (BD Biosciences, Santa Clara, CA, USA) and their suppressive activities against T cells from WT mice was assayed as above.

Western blot analysis

A total of 5×106 various treated CD4+ cells were lysed in RIPA buffer. Specific proteins were detected by immunobloting following the method published previously 39. Briefly, equal amounts (10 μg) of total cell lysate proteins were separated in NuPAGE 4–12% gradient SDS-PAGE gels using a MOP buffering system (Invitrogen). After protein was transferred into PVDF membrane, proteins of interest were detected by specific antibodies.

Real-time RT-PCR

Total cellular RNA were isolated from snap-frozen lung tissue using RNeasy kit (Qiagen, Valencia, CA, USA). The quality was checked by Experion automated electrophoresis system using Experion RNA HighSens analysis kit (Bio-Rad Laboratories, Hercules, CA, USA). Synthesis of cDNA and quantitative RT-PCR analysis were performed using iScript cDNA Synthesis Kit and SYBR Green I dye on iCycler-iQ system (Bio-Rad Laboratories), as reported previously (39). The PCR primers for Foxp3, Bcl-2 and β-actin are: Foxp3, 5′-ACT GGG GTC TTC TCC CTC AA-3′, 5′-CGT GGG AAG GTG CAG AGT AG-3′; Bcl-2, 5′-CCT GGC TGT CTC TGA AGA CC-3′ Bcl2-R: 5′-CTC ACT TGT GGC CCA GGT AT-3′; β-actin: 5′-TGA CAG GAT GCA GAA GGA GA-3′, 5′-GTA CTT GCG CTC AGG AGG AG-3′, respectively. β-actin was used to normalize equal loading of template cDNA.

Statistical analysis

Statistical comparison between various groups was performed by the t-test using GraphPad PRISM software (GraphPad, San Diego, CA, USA). p<0.05 was considered significant.

Acknowledgements

This work was supported by Grant NIH R01 (HL068597), ACR Research and Education Foundation's Within Our Reach, Arthritis Foundation, Outstanding Youth Scientist Investigator Award from National Natural Science Foundation of China (30728007), Webb Foundation and Key Project of Medical Leading Talents of Jiangsu Province in China (2007-2-07), and Zhejiang Province Natural Science Foundation of China (2090918).

Conflict of interest: D. A. Horwitz is a consultant for Becton Dickinson Bioscience, San Jose, CA, USA. The remaining authors declare no financial or commercial conflict of interest.

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